The overall objective of this project is to use hemoglobins found in arcid clams to explore atomic-level structural principles involved in protein cooperativity. The simplicity of these hemoglobins and the diffracting power of their crystals makes them ideal systems for investigating the correlation between structure and function at communicating subunit interfaces. The central role of cooperativity in biological function suggests that principles obtained from such a study will have wide ranging applications to other physiologically important systems. The proposed experiments rely largely on x-ray crystallography and atomic model analysis, but also include thermodynamic experiments on the novel association of subunits in the dimeric hemoglobin from the arcid clam Scapharca inaequivalvis. The crystal structures of the Scapharca dimeric hemoglobin will be refined to a resolution of l.5 Angstroms or better for both the unliganded and CO-liganded states. Additionally, crystals of oxygenated Scapharca dimeric hemoglobin will be grown and subjected to high resolution diffraction analysis. These structures will allow an atomic-level description of the transitions that occur upon ligand binding and lead to cooperative oxygen binding. From lower resolution crystal structures, it appears that cooperativity may result from the dimeric assemblage increasing the intrinsic affinity of the oxygen binding site. To test this hypothesis, the difference between the free energy of association of Scapharca dimeric hemoglobin in the liganded and unliganded states will be determined by sedimentation equilibrium and gel chromatography. Efforts are being made by others to prepare modified dimeric hemoglobins through reconstitution with altered hemes and site-- directed mutagenesis. The crystal structures of various modified hemoglobins will be determined to assess the roles played by the hemes and individual amino acid residues in the cooperative mechanism. Crystallization of a 43OkDa hemoglobin from a related arcid clam, Barbatia reeveana, will be pursued to determine the manner by which a tandem repeat of globin folds can be accommodated while still maintaining cooperative oxygen binding.